Colorectal cancer (CRC) is a major cause of cancer-related morbidity and mortality in the US. Currently, it is thought that sporadic cases of CRC arise when mutations in the APC tumor suppressor/-catenin pathway occur in stem cells. Treatments for metastatic CRC largely target cells undergoing division, including healthy cells, resulting in side effects that greatly limit dose and, consequently, efficacy. Unfortunately, these treatments are also rarely curative. There is considerable hope, however, that selective targeting of tumor- sustaining stem cell populations will induce fewer side effects and improve likelihood of complete responses. The immune system has an exquisite capacity for distinguishing subtle differences between antigens, like the differences between normal cells and cancer cells. Although cancer normally suppresses the immune system, this suppression can be overcome through immunotherapy. In the proposed experiments, we will utilize an immunologic activation mechanism of previously unrecognized potency for treatment of CRC. This mechanism is initiated by IgG binding to tumor cells to form immune complexes (ICs) that potently activate dendritic cells (DCs) to induce T cell-mediated tumor immunity. This mechanism of IC-activated immunity was discovered in our laboratory during studies investigating rejection of engrafted allogeneic tumors. We found that this IC- activated immunity could be applied therapeutically to treat melanoma in a clinically relevant genetically engineered mouse model, as well as various epithelial cancer cell lines including breast, lung, pancreas. The purpose of Aim 1 is to assess one therapeutic strategy using IC-activated DCs to target the immune system against colon cancer stem cells. IC-activated DC vaccination using tumor stem cells will be compared with vaccination using bulk tumor cells. Tumor-bearing mice will be administered DCs and monitored by colonoscopy for responses to therapy. Aim 2 describes an independent strategy for using IC-activated DCs to target tumor stem cells. First, RNA sequencing will be performed to determine changes in the transcriptome of tumor stem cells. Both mutated and aberrantly expressed genes in tumor stem cells will be subject to T cell epitope prediction algorithms. The peptides constituting altered or overexpressed T cell epitopes in total tumor cells, as well as in tumor stem cell populations, will be chemically crosslinked wit IgG to form ICs. These artificial peptide-ICs will be used to activate DCs against desired target peptides in order to stimulate target peptide-recognizing T cells. We expect that in both Aims, targeting tumor stem cells rather than bulk tumor cells will confer additional therapeutic benefit. The strategy developed in Aim 2 will also potentially help enable a novel, personalized immunotherapy for cancer.